RESUMO
Molecular signals interact in networks to mediate biological processes. To analyze these networks, it would be useful to image many signals at once, in the same living cell, using standard microscopes and genetically encoded fluorescent reporters. Here, we report temporally multiplexed imaging (TMI), which uses genetically encoded fluorescent proteins with different clocklike properties-such as reversibly photoswitchable fluorescent proteins with different switching kinetics-to represent different cellular signals. We linearly decompose a brief (few-second-long) trace of the fluorescence fluctuations, at each point in a cell, into a weighted sum of the traces exhibited by each fluorophore expressed in the cell. The weights then represent the signal amplitudes. We use TMI to analyze relationships between different kinase activities in individual cells, as well as between different cell-cycle signals, pointing toward broad utility throughout biology in the analysis of signal transduction cascades in living systems.
Assuntos
Proteínas , Transdução de Sinais , Animais , Humanos , Camundongos , Linhagem Celular , Corantes Fluorescentes , Microscopia de Fluorescência/métodos , Fosforilação , Sobrevivência CelularRESUMO
Genetically encoded fluorescent biosensors are powerful tools for monitoring biochemical activities in live cells, but their multiplexing capacity is limited by the available spectral space. We overcome this problem by developing a set of barcoding proteins that can generate over 100 barcodes and are spectrally separable from commonly used biosensors. Mixtures of barcoded cells expressing different biosensors are simultaneously imaged and analyzed by deep learning models to achieve massively multiplexed tracking of signaling events. Importantly, different biosensors in cell mixtures show highly coordinated activities, thus facilitating the delineation of their temporal relationship. Simultaneous tracking of multiple biosensors in the receptor tyrosine kinase signaling network reveals distinct mechanisms of effector adaptation, cell autonomous and non-autonomous effects of KRAS mutations, as well as complex interactions in the network. Biosensor barcoding presents a scalable method to expand multiplexing capabilities for deciphering the complexity of signaling networks and their interactions between cells.
Assuntos
Técnicas Biossensoriais/métodos , Células/ultraestrutura , Microscopia de Fluorescência/métodos , Análise de Célula Única/métodos , Linhagem Celular Tumoral , HumanosRESUMO
The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.
Assuntos
Carcinogênese/metabolismo , Carcinoma Hepatocelular/genética , Compartimento Celular/genética , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas de Choque Térmico HSP40/genética , Neoplasias Hepáticas/genética , Transdução de Sinais , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinoma Hepatocelular/metabolismo , Compartimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , AMP Cíclico/farmacologia , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Camundongos , Oncogenes/genética , Domínios Proteicos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão , Espectroscopia de Infravermelho com Transformada de Fourier , Imagem com Lapso de Tempo/métodosRESUMO
In order to analyze how a signal transduction network converts cellular inputs into cellular outputs, ideally one would measure the dynamics of many signals within the network simultaneously. We found that, by fusing a fluorescent reporter to a pair of self-assembling peptides, it could be stably clustered within cells at random points, distant enough to be resolved by a microscope but close enough to spatially sample the relevant biology. Because such clusters, which we call signaling reporter islands (SiRIs), can be modularly designed, they permit a set of fluorescent reporters to be efficiently adapted for simultaneous measurement of multiple nodes of a signal transduction network within single cells. We created SiRIs for indicators of second messengers and kinases and used them, in hippocampal neurons in culture and intact brain slices, to discover relationships between the speed of calcium signaling, and the amplitude of PKA signaling, upon receiving a cAMP-driving stimulus.
Assuntos
Corantes Fluorescentes/metabolismo , Genes Reporter , Imagem Óptica , Transdução de Sinais , Animais , Cálcio/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Hipocampo/metabolismo , Humanos , Camundongos , Neurônios/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Células Piramidais/metabolismoRESUMO
Transcriptional regulation in metazoans occurs through long-range genomic contacts between enhancers and promoters, and most genes are transcribed in episodic "bursts" of RNA synthesis. To understand the relationship between these two phenomena and the dynamic regulation of genes in response to upstream signals, we describe the use of live-cell RNA imaging coupled with Hi-C measurements and dissect the endogenous regulation of the estrogen-responsive TFF1 gene. Although TFF1 is highly induced, we observe short active periods and variable inactive periods ranging from minutes to days. The heterogeneity in inactive times gives rise to the widely observed "noise" in human gene expression and explains the distribution of protein levels in human tissue. We derive a mathematical model of regulation that relates transcription, chromosome structure, and the cell's ability to sense changes in estrogen and predicts that hypervariability is largely dynamic and does not reflect a stable biological state.
Assuntos
Regulação da Expressão Gênica/fisiologia , Expressão Gênica/fisiologia , Transcrição Gênica/fisiologia , Receptor alfa de Estrogênio/metabolismo , Estrogênios , Expressão Gênica/genética , Humanos , Modelos Teóricos , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/metabolismo , Análise de Célula Única/métodos , Transcrição Gênica/genética , Ativação Transcricional/fisiologia , Fator Trefoil-1/genéticaRESUMO
The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the leading target for next-generation vaccines against the disease-causing blood-stage of malaria. However, little is known about how human antibodies confer functional immunity against this antigen. We isolated a panel of human monoclonal antibodies (mAbs) against PfRH5 from peripheral blood B cells from vaccinees in the first clinical trial of a PfRH5-based vaccine. We identified a subset of mAbs with neutralizing activity that bind to three distinct sites and another subset of mAbs that are non-functional, or even antagonistic to neutralizing antibodies. We also identify the epitope of a novel group of non-neutralizing antibodies that significantly reduce the speed of red blood cell invasion by the merozoite, thereby potentiating the effect of all neutralizing PfRH5 antibodies as well as synergizing with antibodies targeting other malaria invasion proteins. Our results provide a roadmap for structure-guided vaccine development to maximize antibody efficacy against blood-stage malaria.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antiprotozoários/imunologia , Eritrócitos/parasitologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Adolescente , Adulto , Animais , Sítios de Ligação , Proteínas de Transporte/imunologia , Reações Cruzadas/imunologia , Epitopos/imunologia , Feminino , Células HEK293 , Voluntários Saudáveis , Humanos , Malária Falciparum/parasitologia , Masculino , Merozoítos/fisiologia , Pessoa de Meia-Idade , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/imunologia , Coelhos , Ratos , Ratos Sprague-Dawley , Adulto JovemRESUMO
The immune system encodes information about the severity of a pathogenic threat in the quantity and type of memory cells it forms. This encoding emerges from lymphocyte decisions to maintain or lose self-renewal and memory potential during a challenge. By tracking CD8+ T cells at the single-cell and clonal lineage level using time-resolved transcriptomics, quantitative live imaging, and an acute infection model, we find that T cells will maintain or lose memory potential early after antigen recognition. However, following pathogen clearance, T cells may regain memory potential if initially lost. Mechanistically, this flexibility is implemented by a stochastic cis-epigenetic switch that tunably and reversibly silences the memory regulator, TCF1, in response to stimulation. Mathematical modeling shows how this flexibility allows memory T cell numbers to scale robustly with pathogen virulence and immune response magnitudes. We propose that flexibility and stochasticity in cellular decisions ensure optimal immune responses against diverse threats.
Assuntos
Linfócitos T CD8-Positivos , Células T de Memória , Epigênese Genética , Células Clonais , Memória Imunológica , Diferenciação CelularRESUMO
Transcription coactivators are proteins or protein complexes that mediate transcription factor (TF) function. However, they lack DNA-binding capacity, prompting the question of how they engage target loci. Three non-exclusive hypotheses have been posited: coactivators are recruited by complexing with TFs, by binding histones through epigenetic reader domains, or by partitioning into condensates through their extensive intrinsically disordered regions. Using p300 as a prototypical coactivator, we systematically mutated its annotated domains and show by single-molecule tracking in live U2OS cells that coactivator-chromatin binding depends entirely on combinatorial binding of multiple TF-interaction domains. Furthermore, we demonstrate that acetyltransferase activity opposes p300-chromatin association and that the N-terminal TF-interaction domains regulate that activity. Single TF-interaction domains are insufficient for chromatin binding and regulation of catalytic activity, implying a principle that we speculate could broadly apply to eukaryotic gene regulation: a TF must act in coordination with other TFs to recruit coactivator activity.
Assuntos
Fatores de Transcrição , Transcrição Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Cromatina/genéticaRESUMO
RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , RNA Polimerase II , Transcrição Gênica , Fatores de Elongação da Transcrição , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Fator B de Elongação Transcricional Positiva/genética , Regiões Promotoras Genéticas , Sistemas CRISPR-Cas , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Cromossomos Politênicos/genética , Cromossomos Politênicos/metabolismo , Regulação da Expressão Gênica , Fosforilação , Ligação Proteica , Resposta ao Choque Térmico/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Nucleossomos/metabolismo , Nucleossomos/genéticaRESUMO
Metabolic enzymes can adapt during energy stress, but the consequences of these adaptations remain understudied. Here, we discovered that hexokinase 1 (HK1), a key glycolytic enzyme, forms rings around mitochondria during energy stress. These HK1-rings constrict mitochondria at contact sites with the endoplasmic reticulum (ER) and mitochondrial dynamics protein (MiD51). HK1-rings prevent mitochondrial fission by displacing the dynamin-related protein 1 (Drp1) from mitochondrial fission factor (Mff) and mitochondrial fission 1 protein (Fis1). The disassembly of HK1-rings during energy restoration correlated with mitochondrial fission. Mechanistically, we identified that the lack of ATP and glucose-6-phosphate (G6P) promotes the formation of HK1-rings. Mutations that affect the formation of HK1-rings showed that HK1-rings rewire cellular metabolism toward increased TCA cycle activity. Our findings highlight that HK1 is an energy stress sensor that regulates the shape, connectivity, and metabolic activity of mitochondria. Thus, the formation of HK1-rings may affect mitochondrial function in energy-stress-related pathologies.
Assuntos
Dinaminas , Metabolismo Energético , Hexoquinase , Mitocôndrias , Dinâmica Mitocondrial , Proteínas Mitocondriais , Hexoquinase/metabolismo , Hexoquinase/genética , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/genética , Mitocôndrias/enzimologia , Dinaminas/metabolismo , Dinaminas/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Animais , Trifosfato de Adenosina/metabolismo , Estresse Fisiológico , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Ciclo do Ácido Cítrico , Glucose-6-Fosfato/metabolismo , Camundongos , Células HeLa , Células HEK293 , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , MutaçãoRESUMO
Telomerase maintains genome integrity by adding repetitive DNA sequences to the chromosome ends in actively dividing cells, including 90% of all cancer cells. Recruitment of human telomerase to telomeres occurs during S-phase of the cell cycle, but the molecular mechanism of the process is only partially understood. Here, we use CRISPR genome editing and single-molecule imaging to track telomerase trafficking in nuclei of living human cells. We demonstrate that telomerase uses three-dimensional diffusion to search for telomeres, probing each telomere thousands of times each S-phase but only rarely forming a stable association. Both the transient and stable association events depend on the direct interaction of the telomerase protein TERT with the telomeric protein TPP1. Our results reveal that telomerase recruitment to telomeres is driven by dynamic interactions between the rapidly diffusing telomerase and the chromosome end.
Assuntos
Telomerase/metabolismo , Telômero/enzimologia , Transporte Ativo do Núcleo Celular , Proteínas de Bactérias , Proteína 9 Associada à CRISPR , Linhagem Celular , Núcleo Celular/enzimologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Corpos Enovelados/enzimologia , Endonucleases , Edição de Genes , Genoma Humano , Células HeLa , Humanos , Imageamento Tridimensional , Domínios Proteicos , Fase S , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Complexo Shelterina , Telomerase/química , Telômero/química , Homeostase do Telômero , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/metabolismoRESUMO
DNA supercoiling has emerged as a major contributor to gene regulation in bacteria, but how DNA supercoiling impacts transcription dynamics in eukaryotes is unclear. Here, using single-molecule dual-color nascent transcription imaging in budding yeast, we show that transcriptional bursting of divergent and tandem GAL genes is coupled. Temporal coupling of neighboring genes requires rapid release of DNA supercoils by topoisomerases. When DNA supercoils accumulate, transcription of one gene inhibits transcription at its adjacent genes. Transcription inhibition of the GAL genes results from destabilized binding of the transcription factor Gal4. Moreover, wild-type yeast minimizes supercoiling-mediated inhibition by maintaining sufficient levels of topoisomerases. Overall, we discover fundamental differences in transcriptional control by DNA supercoiling between bacteria and yeast and show that rapid supercoiling release in eukaryotes ensures proper gene expression of neighboring genes.
Assuntos
Saccharomyces cerevisiae , Transcrição Gênica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , DNA Topoisomerases Tipo II/genética , DNA , DNA Bacteriano/genética , DNA Super-Helicoidal/genética , DNA Topoisomerases Tipo I/metabolismoRESUMO
Macrophages initiate inflammatory responses via the transcription factor NFκB. The temporal pattern of NFκB activity determines which genes are expressed and thus, the type of response that ensues. Here, we examined how information about the stimulus is encoded in the dynamics of NFκB activity. We generated an mVenus-RelA reporter mouse line to enable high-throughput live-cell analysis of primary macrophages responding to host- and pathogen-derived stimuli. An information-theoretic workflow identified six dynamical features-termed signaling codons-that convey stimulus information to the nucleus. In particular, oscillatory trajectories were a hallmark of responses to cytokine but not pathogen-derived stimuli. Single-cell imaging and RNA sequencing of macrophages from a mouse model of Sjögren's syndrome revealed inappropriate responses to stimuli, suggestive of confusion of two NFκB signaling codons. Thus, the dynamics of NFκB signaling classify immune threats through six signaling codons, and signal confusion based on defective codon deployment may underlie the etiology of some inflammatory diseases.
Assuntos
Códon/genética , Macrófagos/fisiologia , NF-kappa B/genética , Transdução de Sinais/genética , Animais , Células Cultivadas , Citocinas/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Inflamação/genética , Camundongos , Camundongos Endogâmicos C57BL , Síndrome de Sjogren/genética , Fator de Transcrição RelA/genéticaRESUMO
Owing to their unique abilities to manipulate, label, and image individual molecules in vitro and in cellulo, single-molecule techniques provide previously unattainable access to elementary biological processes. In imaging, single-molecule fluorescence resonance energy transfer (smFRET) and protein-induced fluorescence enhancement in vitro can report on conformational changes and molecular interactions, single-molecule pull-down (SiMPull) can capture and analyze the composition and function of native protein complexes, and single-molecule tracking (SMT) in live cells reveals cellular structures and dynamics. In labeling, the abilities to specifically label genomic loci, mRNA, and nascent polypeptides in cells have uncovered chromosome organization and dynamics, transcription and translation dynamics, and gene expression regulation. In manipulation, optical tweezers, integration of single-molecule fluorescence with force measurements, and single-molecule force probes in live cells have transformed our mechanistic understanding of diverse biological processes, ranging from protein folding, nucleic acids-protein interactions to cell surface receptor function.
Assuntos
Genômica/tendências , Imagem Molecular/tendências , Imagem Óptica/tendências , Imagem Individual de Molécula/tendências , Animais , Difusão de Inovações , Transferência Ressonante de Energia de Fluorescência/tendências , Humanos , Microscopia de Fluorescência/tendências , Proteômica/tendênciasRESUMO
Transcription initiation by RNA polymerase II (RNA Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus, where thousands of promoters are broadly distributed in chromatin, it is unclear how multiple individual components converge on any target to establish the PIC. Here we use live-cell, single-molecule tracking in S. cerevisiae to visualize constrained exploration of the nucleoplasm by PIC components and Mediator's key role in guiding this process. On chromatin, TFIID/TATA-binding protein (TBP), Mediator, and RNA Pol II instruct assembly of a short-lived PIC, which occurs infrequently but efficiently within a few seconds on average. Moreover, PIC exclusion by nucleosome encroachment underscores regulated promoter accessibility by chromatin remodeling. Thus, coordinated nuclear exploration and recruitment to accessible targets underlies dynamic PIC establishment in yeast. Our study provides a global spatiotemporal model for transcription initiation in live cells.
Assuntos
Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Iniciação da Transcrição Genética/fisiologia , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Complexo Mediador/genética , Nucleossomos/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise Espaço-Temporal , Proteína de Ligação a TATA-Box/genética , Fator de Transcrição TFIID/genética , Transcrição Gênica/genéticaRESUMO
The coactivator p300/CREB-binding protein (CBP) regulates genes by facilitating the assembly of transcriptional machinery and by acetylating histones and other factors. However, it remains mostly unclear how both functions of p300 are dynamically coordinated during gene control. Here, we showed that p300 can orchestrate two functions through the formation of dynamic clusters with certain transcription factors (TFs), which is mediated by the interactions between a TF's transactivation domain (TAD) and the intrinsically disordered regions of p300. Co-condensation can enable spatially defined, all-or-none activation of p300's catalytic activity, priming the recruitment of coactivators, including Brd4. We showed that co-condensation can modulate transcriptional initiation rate and burst duration of target genes, underlying nonlinear gene regulatory functions. Such modulation is consistent with how p300 might shape gene bursting kinetics globally. Altogether, these results suggest an intriguing gene regulation mechanism, in which TF and p300 co-condensation contributes to transcriptional bursting regulation and cooperative gene control.
Assuntos
Proteína p300 Associada a E1A/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Ativação Transcricional/genética , Acetilação , Animais , Células CHO , Proteína de Ligação a CREB/metabolismo , Linhagem Celular , Cricetulus , Regulação da Expressão Gênica/genética , Células HEK293 , Histonas/metabolismo , Humanos , Cinética , Camundongos , Transativadores/metabolismoRESUMO
DNA replication is carried out by a multi-protein machine called the replisome. In Saccharomyces cerevisiae, the replisome is composed of over 30 different proteins arranged into multiple subassemblies, each performing distinct activities. Synchrony of these activities is required for efficient replication and preservation of genomic integrity. How this is achieved is particularly puzzling at the lagging strand, where current models of the replisome architecture propose turnover of the canonical lagging strand polymerase, Pol δ, at every cycle of Okazaki fragment synthesis. Here, we established single-molecule fluorescence microscopy protocols to study the binding kinetics of individual replisome subunits in live S. cerevisiae. Our results show long residence times for most subunits at the active replisome, supporting a model where all subassemblies bind tightly and work in a coordinated manner for extended periods, including Pol δ, redefining the architecture of the active eukaryotic replisome.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Células Eucarióticas/metabolismo , Complexos Multienzimáticos/metabolismo , Núcleo Celular/metabolismo , Cinética , Modelos Biológicos , Proteínas Nucleares/metabolismo , Subunidades Proteicas/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula , Fatores de TempoRESUMO
Biological phase transitions form membrane-less organelles that generate distinct cellular environments. How molecules are partitioned between these compartments and the surrounding cellular space and the functional consequence of this localization is not well understood. Here, we report the localization of mRNA to stress granules (SGs) and processing bodies (PBs) and its effect on translation and degradation during the integrated stress response. Using single mRNA imaging in living human cells, we find that the interactions of mRNAs with SGs and PBs have different dynamics, very few mRNAs directly move between SGs and PBs, and that specific RNA-binding proteins can anchor mRNAs within these compartments. During recovery from stress, we show that mRNAs that were within SGs and PBs are translated and degraded at similar rates as their cytosolic counterparts. Our work provides a framework for using single-molecule measurements to directly investigate the molecular mechanisms of phase-separated compartments within their cellular environment.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Hibridização in Situ Fluorescente , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/metabolismo , Imagem Individual de Molécula/métodos , Estresse Fisiológico , Autoantígenos/genética , Autoantígenos/metabolismo , Transporte Biológico , Grânulos Citoplasmáticos/genética , Células HeLa , Humanos , Microscopia de Fluorescência , Ligação Proteica , Sequência de Oligopirimidina na Região 5' Terminal do RNA , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Fatores de Tempo , Antígeno SS-BRESUMO
Nonsense-mediated decay (NMD) is a surveillance system that degrades mRNAs containing a premature termination codon (PTC) and plays important roles in protein homeostasis and disease. The efficiency of NMD is variable, impacting the clinical outcome of genetic mutations. However, limited resolution of bulk analyses has hampered the study of NMD efficiency. Here, we develop an assay to visualize NMD of individual mRNA molecules in real time. We find that NMD occurs with equal probability during each round of translation of an mRNA molecule. However, this probability is variable and depends on the exon sequence downstream of the PTC, the PTC-to-intron distance, and the number of introns both upstream and downstream of the PTC. Additionally, a subpopulation of mRNAs can escape NMD, further contributing to variation in NMD efficiency. Our study uncovers real-time dynamics of NMD, reveals key mechanisms that influence NMD efficiency, and provides a powerful method to study NMD.
Assuntos
Códon sem Sentido/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , RNA Mensageiro/genética , Códon sem Sentido/química , Éxons/genética , Humanos , Íntrons/genética , Mutação/genética , Estabilidade de RNA/genética , RNA Mensageiro/química , Imagem Individual de MoléculaRESUMO
RNA localization in eukaryotes is a mechanism to regulate transcripts fate. Conversely, bacterial transcripts were not assumed to be specifically localized. We previously demonstrated that E. coli mRNAs may localize to where their products localize in a translation-independent manner, thus challenging the transcription-translation coupling extent. However, the scope of RNA localization in bacteria remained unknown. Here, we report the distribution of the E. coli transcriptome between the membrane, cytoplasm, and poles by combining cell fractionation with deep-sequencing (Rloc-seq). Our results reveal asymmetric RNA distribution on a transcriptome-wide scale, significantly correlating with proteome localization and prevalence of translation-independent RNA localization. The poles are enriched with stress-related mRNAs and small RNAs, the latter becoming further enriched upon stress in an Hfq-dependent manner. Genome organization may play a role in localizing membrane protein-encoding transcripts. Our results show an unexpected level of intricacy in bacterial transcriptome organization and highlight the poles as hubs for regulation.